Collagen-polymer conjugates

ABSTRACT

Collagen, particularly atelopeptide collagen, exhibits improved handling characteristics when chemically conjugated and/or crosslinked with a synthetic hydrophilic polymer.

This application is a continuation-in-part of copending U.S. patentapplication Ser. No. 274,071, filed Nov. 21, 1988 and now abandoned,incorporated herein by reference in full.

DESCRIPTION

1. Technical Field

This invention relates to proteins and chemically-modified proteins.More specifically, this invention relates to collagen modified byconjugation with synthetic hydrophilic polymers.

2. Background of the Invention

Collagen is the major protein component of bone, cartilage, skin, andconnective tissue in animals. Collagen in its native form is typically arigid, rod-shaped molecule approximately 300 nm long and 1.5 nm indiameter. It is composed of three collagen polypeptides which form atight triple helix. The collagen polypeptides are characterized by along midsection having the repeating sequence -Gly-X-Y-, where X and Yare often proline or hydroxyproline, bounded at each end by the"telopeptide" regions, which constitute less than about 5% of themolecule. The telopeptide regions of the collagen chains are typicallyresponsible for the crosslinking between chains, and for theimmunogenicity of the protein. Collagen occurs in several "types",having differing physical properties. The most abundant types are TypesI-III.

Collagen is typically isolated from natural sources, such as bovinehide, cartilage, or bones. Bones are usually dried, defatted, crushed,and demineralized to extract collagen, while hide and cartilage areusually minced and digested with proteolytic enzymes (other thancollagenase). As collagen is resistant to most proteolytic enzymes, thisprocedure conveniently serves to remove most of the contaminatingprotein found with collagen.

Collagen may be denatured by boiling, which produces the familiarproduct gelatin.

Daniels et al, U.S. Pat. No. 3,949,073, disclosed the preparation ofsoluble collagen by dissolving tissue in aqueous acid, followed byenzymatic digestion. The resulting atelopeptide collagen is soluble, andsubstantially less immunogenic than unmodified collagen It may beinjected into suitable locations of a subject with a fibril-formationpromoter (described as a polymerization promoter in the patent) to formfibrous collagen implants in situ, for augmenting hard or soft tissue.This material is now commercially available from Collagen Corporation(Palo Alto, CA) under the trademark Zyderm® collagen implant.

Luck et al, U.S. Pat. No. 4,488,911, disclosed a method for preparingcollagen in solution (CIS), wherein native collagen is extracted fromanimal tissue in dilute aqueous acid, followed by digestion with anenzyme such as pepsin, trypsin, or Pronase®. The enzyme digestionremoves the telopeptide portions of the collagen molecules, providing"atelopeptide" collagen in solution. The atelopeptide CIS so produced issubstantially nonimmunogenic, and is also substantially non-cross-linkeddue to loss of the primary crosslinking regions. The CIS may then beprecipitated by dialysis in a moderate shear environment to producecollagen fibers which resemble native collagen fibers. The precipitated,reconstituted fibers may additionally be crosslinked using a chemicalagent (for example aldehydes such as formaldehyde and glutaraldehyde),or using heat or radiation. The resulting products are suitable for usein medical implants due to their biocompatability and reducedimmunogenicity.

Wallace et al, U.S. Pat. No. 4,424,208, disclosed an improved collagenformulation suitable for use in soft tissue augmentation. Wallace'sformulation comprises reconstituted fibrillar atelopeptide collagen (forexample, Zyderm® collagen) in combination with particulate, crosslinkedatelopeptide collagen dispersed in an aqueous medium. The addition ofparticulate crosslinked collagen improves the implant's persistence, orability to resist shrinkage following implantation.

Smestad et al, U.S. Pat. No. 4,582,640, disclosed a glutaraldehydecrosslinked atelopeptide CIS preparation (GAX) suitable for use inmedical implants. The collagen is cross-linked under conditions favoringintrafiber bonding rather than interfiber bonding, and provides aproduct with higher persistence than non-cross-linked atelopeptidecollagen, and is commercially available from Collagen Corporation underthe trademark Zyplast® Implant.

Nguyen et al, U.S. Pat. No. 4,642,117, disclosed a method for reducingthe viscosity of atelopeptide CIS by mechanical shearing Reconstitutedcollagen fibers are passed through a fine-mesh screen until viscosity isreduced to a practical level for injection.

Nathan et al, U.S. Pat. No. 4,563,350, disclosed osteoinductive bonerepair compositions comprising an osteo-inductive factor, at least 5%nonreconstituted (afibrillar) collagen, and the remainder reconstitutedcollagen and/or mineral powder (e.g., hydroxyapatite) CIS may be usedfor the nonreconstituted collagen, and Zyderm® collagen implant (ZCI) ispreferred for the reconstituted collagen component. The material isimplanted in bone defects or fractures to speed ingrowth of osteoclastsand promote new bone growth.

Chu, U.S. Pat. No. 4,557,764, disclosed a "second nucleation" collagenprecipitate which exhibits a desirable malleability and putty-likeconsistency. Collagen is provided in solution (e.g., at 2-4 mg/mL), anda "first nucleation product" is precipitated by rapid titration andcentrifugation. The remaining supernatant (containing the bulk of theoriginal collagen) is then decanted and allowed to stand overnight. Theprecipitated second nucleation product is collected by centrifugation.

Chu, U.S. Pat. No. 4,689,399, disclosed a collagen membrane preparation,which is prepared by compressing and drying a collagen gel. Theresulting product has high tensile strength.

J. A. M. Ramshaw et al, Anal Biochem (1984) 141:361-65, and PCTapplication W087/04078 disclosed the precipitation of bovine collagen(types I, II, and III) from aqueous PEG solutions, where there is nobinding between collagen and PEG.

Werner, U.S. Pat. No. 4,357,274, disclosed a method for improving thedurability of sclero protein (e.g., brain meninges) by soaking thedegreased tissue in H₂ O₂ or PEG for several hours prior tolyophilizing. The resulting modified whole tissue exhibits increasedpersistence.

Hiroyoshi, U.S. Pat. No 4,678,468, disclosed the preparation ofpolysiloxane polymers having an interpenetrating network ofwater-soluble polymer dispersed within. The water-soluble polymer may bea collagen derivative, and the polymer may additionally include heparin.The polymers are shaped into artificial blood vessel grafts, and aredesigned to prevent clotting.

Other patents disclose the use of collagen preparations with bonefragments or minerals. For example, Miyata et al, U.S. Pat. No.4,314,380 disclosed a bone implant prepared by baking animal bonesegments, and soaking the baked segments in a solution of atelopeptidecollagen. Deibig et al, U.S. Pat. No. 4,192,021 disclosed an implantmaterial which comprises powdered calcium phosphate in a pastyformulation with a biodegradable polymer (which may be collagen).Commonly-owned copending U.S. patent application Ser. No. 855,004, filedApr. 22, 1986, disclosed a particularly effective bone repair materialcomprising autologous bone marrow, collagen, and particulate calciumphosphate in a solid, malleable formulation.

There are several references in the art to proteins modified by covalentconjugation to polymers, to alter the solubility, antigenicity andbiological clearance of the protein For example, U.S. Pat. No. 4,261,973disclosed the conjugation of several allergans to PEG or PPG(polypropylene glycol) to reduce the proteins, immunogenicity. U.S. Pat.No. 4,301,144 disclosed the conjugation of hemoglobin with PEG and otherpolymers to increase the protein's oxygen carrying capability. EPO98,110 disclosed coupling an enzyme or interferon to apolyoxyethylene-polyoxypropylene (POE-POP) block polymer increases theprotein's halflife in serum. U.S. Pat. No. 4,179,337 disclosedconjugating hydrophilic enzymes and insulin to PEG or PPG to reduceimmunogenicity. Davis et al, Lancet (1981) 2:281-83 disclosed the enzymeuricase modified by conjugation with PEG to provide uric acid metabolismin serum having a long halflife and low immunogenicity. Nishida et al, JPharm Pharmacol (1984) 36:354-55 disclosed PEG-uricase conjugatesadministered orally to chickens, demonstrating decreased serum levels ofuric acid. Inada et al, Biochem & Biophys Res Comm (1984) 122:845-50disclosed lipoprotein lipase conjugation with PEG to render it solublein organic solvents. Takahashi et al, Biochem & Biophys Res Comm (1984)121:261-65 disclosed HRP conjugated with PEG to render the enzymesoluble in benzene. Abuchowski et al, Cancer Biochem Biophys (1984)7:175-86 disclosed that enzymes such as asparaginase, catalase, uricase,arginase, trypsin, superoxide dismutase, adenosine deaminase,phenylalanine ammonia-lyase, and the like, conjugated with PEG exhibitlonger half-lives in serum and decreased immunogenicity. However, thesereferences are essentially concerned with modifying the solubility andbiological characteristics of proteins administered in lowconcentrations in aqueous solution.

M. Chvapil et al, J Biomed Mater Res (1969) 3:315-32 disclosed acomposition prepared from collagen sponge and a crosslinked ethyleneglycol monomethacrylate-ethylene glycol dimethacrylate hydrogel. Thecollagen sponge was prepared by lyophilizing an aqueous mixture ofbovine hide collagen and methylglyoxal (a tanning agent). Thesponge-hydrogel composition was prepared by polymerizing ethylene glycolmonomethacrylate and ethylene glycol dimethacrylate in the sponge.

DISCLOSURE OF THE INVENTION

We have discovered that formulations containing reconstituted fibrillaratelopeptide collagen in combination with particulate mineral components(useful, e.g., for treating bone defects and fractures) exhibit physicalinstability with time, and tend to separate into several phases orlayers. Further, the handling characteristics of such compositions arenot ideal, and the malleability and elasticity of such formulationscould be improved.

We have now invented a new collagen-polymer conjugate which exhibitssuperior handling and chemical stability characteristics. The collagen,preferably reconstituted atelopeptide collagen, is chemically bonded toa synthetic hydrophilic polymer, preferably polyethylene glycol, to forma new collagen-polymer conjugate.

The polymer may be monofunctional or polyfunctional, having one endcapable of attachment, or two or more ends capable of attachment. Whenthe polymer is polyfunctional, it may be joined to collagen by one ormore ends, i.e., the polymer may crosslink collagen molecules. Thecollagen-polymer conjugates may be used to replace or reinforce softtissue, and may be used in combination with a suitable particulatematerial to treat bone defects. These materials are also useful forcoating implants (such as catheters and bone implants) to reduceimmunogenicity and foreign body reactions. Dried collagen-polymerconjugates, cast into a membranous form, may be used to replace orrepair damaged skin (e.g., burned skin), nerve sheaths, blood vessels,heart valves, ophthalmic shields and corneal lenticules. These forms mayalso be used in dental applications (e.g. for guided tissueregeneration).

The crosslinking reaction between the collagen and polymer may beperformed in vitro, or a reaction mixture may be injected forcrosslinking in situ. At sufficient density, crosslinkedcollagen-polymer conjugates resemble cartilage, and are useful assubstitutes therefor, (e.g. cranial onlay, ear and nose reconstruction,and the like). Polyfunctional polymers may also be used to crosslinkcollagen molecules to other proteins (e.g., glycosaminoglycans,chondroitin sulfates, fibronectin, and the like), particularly growthfactors, for compositions particularly suited for wound healing,osteogenesis, and immune modulation. Such tethering of growth factors tocollagen molecules provides an effective slow-release drug deliverysystem.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the force necessary to extrude three compositions:Zyderm® collagen implant (ZCI), a glutaraldehyde-crosslinked collagen(GAX), and a collagen-PEG conjugate of the invention.

FIG. 2 illustrates the results of the experiment conducted in Example6E, demonstrating the retention of biologically active TGF-β1 in acrosslinked collagen-dPEG composition.

MODES OF CARRYING OUT THE INVENTION A. Definitions

The term "collagen" as used herein refers to all forms of collagen,including those which have been processed or otherwise modified.Preferred collagens are treated to remove the immunogenic telopeptideregions ("atelopeptide collagen"), are soluble, and will have beenreconstituted into fibrillar form. Type I collagen is best suited tomost applications involving bone or cartilage repair. However, otherforms of collagen are also useful in the practice of the invention, andare not excluded from consideration here. Collagen cross-linked usingheat, radiation, or chemical agents such as glutaraldehyde may beconjugated with polymers as described herein to form particularly rigidcompositions. Collagen crosslinked using glutaraldehyde or other(nonpolymer) linking agents is referred to herein as "GAX", whilecollagen cross-linked using heat and/or radiation is termed "HRX."0

The term "synthetic hydrophilic polymer" as used herein refers to asynthetic polymer having an average molecular weight and compositionwhich renders the polymer essentially water-soluble. Most hydrophilicpolymers achieve this property by incorporating a sufficient number ofoxygen (or less frequently nitrogen) atoms available for forminghydrogen bonds in aqueous solution Hydrophilic polymers used herein willgenerally be polyoxyethylene, polyethylene glycol, polymethylene glycol,polytrimethylene glycols, polyvinylpyrrolidones, or derivatives thereof.The polymers are preferably linear or only slightly branched (i.e.,having only about 2-10 significant free ends), and will not besubstantially cross-linked. Other suitable polymers includepolyoxyethylene-polyoxypropylene block polymers and copolymers.Polyoxyethylene-polyoxypropylene block polymers having an ethylenediamine nucleus (and thus having four ends) are also available and maybe used in the practice of the invention. Naturally occurring polymerssuch as proteins, starch, cellulose, heparin and the like are expresslyexcluded from the scope of this definition. All suitable polymers willbe non-toxic and non-inflammatory when administered subcutaneously, andwill preferably be essentially nondegradable in vivo over a period of atleast several months. The hydrophilic polymer may increase thehydrophilicity of the collagen, but does not render it water soluble.Presently preferred hydrophilic polymers are mono- and difunctionalpolyethylene glycols (PEG). Monofunctional PEG has only one reactivehydroxy group, while difunctional PEG preferably has reactive groups ateach end. Monofunctional PEG preferably has an average molecular weightbetween about 300 and about 15,000, more preferably between about 1,900and about 8,000, and most preferably about 5,000. Difunctional PEGpreferably has a molecular weight of about 400 to about 20,000, morepreferably about 3,000 to about 10,000. PEG can be renderedmonofunctional by forming an alkylene ether at one end. The alkyleneether may be any suitable alkoxy radical having 1-6 carbon atoms, forexample, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, hexyloxy, and thelike. Methoxy is presently preferred. Difunctional PEG is provided byallowing a reactive hydroxy group at each end of the linear molecule.The reactive groups are preferably at the ends of the polymer, but maybe provided along the length thereof. Polyfunctional molecules arecapable of crosslinking the compositions of the invention, and may beused to attach biological growth factors to collagen.

The term "chemically conjugated" as used herein means attached through acovalent chemical bond. In the practice of the invention, a synthetichydrophilic polymer and collagen may be chemically conjugated by using alinking radical, so that the polymer and collagen are each bound to theradical, but not directly to each other. The term "collagen-polymer"refers to collagen chemically conjugated to a synthetic hydrophilicpolymer, within the meaning of this invention. Thus, "collagen-PEG" (or"PEG-collagen") denotes a composition of the invention wherein collagenis chemically conjugated to PEG. "Collagen-dPEG" refers to collagenchemically conjugated to difunctional PEG, wherein the collagenmolecules are typically crosslinked. "Crosslinked collagen" refers tocollagen in which collagen molecules are linked by covalent bonds withpolyfunctional (including difunctional) polymers. Terms such as"GAX-dPEG" and "HRX-dPEG" indicate collagen crosslinked by both adifunctional hydrophilic polymer and a crosslinking agent such asglutaraldehyde or heat.

Those of ordinary skill in the art will appreciate that syntheticpolymers such as polyethyleneglycol cannot practically be preparedhaving exact molecular weights, and that the term "molecular weight" asused herein refers to the average molecular weight of a number ofmolecules in any given sample, as commonly used in the art. Thus, asample of PEG 2,000 might contain polymer molecules ranging in weightfrom, for example, 1,200 to 2,500 daltons. Specification of a range ofmolecular weight indicates that the average molecular weight may be anyvalue between the limits specified, and may include molecules outsidethose limits. Thus, a molecular weight range of about 800 to about20,000 indicates an average molecular weight of at least about 800,ranging up to about 20 kDa.

The term "available lysine residue" as used herein refers to lysine sidechains exposed on the outer surface of collagen molecules, which arepositioned in a manner allowing reaction with activated PEG. The numberof available lysine residues may be determined by reaction with sodium2,4,6-trinitrobenzenesulfonate (TNBS).

The terms "treat" and "treatment" as used herein refer to augmentation,repair, prevention, or alleviation of defects, particularly defects dueto loss or absence of soft tissue or soft tissue support, or to loss orabsence of bone. Additionally, "treat" and "treatment" also refer to theprevention, maintenance, or alleviation of disorders or disease using abiologically active protein coupled to the collagen-polymer compositionof the invention. Accordingly, treatment of soft tissue includesaugmentation of soft tissue, for example implantation ofcollagen-polymer conjugates of the invention to restore normal ordesirable dermal contours, as in the removal of dermal creases orfurrows, or as in the replacement of subcutaneous fat in maxillary areaswhere the fat is lost due to aging. Treatment of bone and cartilageincludes the use of collagen-polymer conjugates, and particularlycollagen-PEG in combination with suitable particulate materials, toreplace or repair bone tissue, for example in the treatment of bonenonunions or fractures. Treatment of bone also includes use ofcartilaginoid collagen-dPEG compositions, with or without additionalbone growth factors. Compositions comprising collagen-polymer withceramic particles, preferably hydroxyapatite and/or tricalciumphosphate, are particularly useful for the repair of stress-bearing bonedue to its high tensile strength. Compositions of the invention mayadditionally include biologically active factors to aid in healing orregrowth of normal tissue. For example, one may incorporate factors suchas epidermal growth factor (EGF), transforming growth factor (TGF)alpha, TGF-β (including any combination of TGF-βs), TGF-β1, TGF-β2,platelet derived growth factor (PDGF-AA, PDGF-AB, PDGF-BB), acidicfibroblast growth factor (FGF), basic FGF, connective tissue activatingpeptides (CTAP), β-thromboglobulin, insulin-like growth factors, tumornecrosis factors (TNF), interleukins, colony stimulating factors (CSFs),erythropoietin (EPO), nerve growth factor (NGF), interferons (IFN),osteogenic factors, and the like. Incorporation of such factors, andappropriate combinations of factors, can facilitate the regrowth andremodeling of the implant into normal bone tissue, or may be used in thetreatment of wounds. Further, one may chemically link the factors to thecollagen-polymer composition by employing a suitable amount ofpolyfunctional polymer molecules during synthesis. The factors may thenbe attached to the free polymer ends by the same method used to attachPEG to collagen, or by any other suitable method. By tethering factormolecules to the implant, the effective amount of factor issubstantially reduced. Dried collagen-PEG compositions havingsponge-like characteristics may be prepared as wound dressings, or whenincorporated with growth factors or the like, they serve as effectivecontrolled-release drug delivery matrices.

The term "effective amount" refers to the amount of composition requiredin order to obtain the effect desired. Thus, a "tissue growth promotingamount" of a composition containing a growth factor refers to the amountof factor needed in order to stimulate tissue growth to a detectabledegree. Tissue, in this context, includes connective tissue, bone,cartilage, epidermis and dermis, blood, and other tissues.

The term "sufficient amount" as used herein is applied to the amount ofcarrier used in combination with the collagen-polymer conjugates of theinvention. A sufficient amount is that amount which when mixed with theconjugate renders it in the physical form desired, for example,injectable solution, injectable suspension, plastic or malleableimplant, rigid stress-bearing implant, and so forth.

The term "suitable particulate material" as used herein refers to aparticulate material which is substantially insoluble in water, which isbiocompatible, and which is immiscible with collagen-polymer. Theparticles of material may be fibrillar, or may range in size from about1 to 20 μm in diameter and be bead-like or irregular in shape. Exemplaryparticulate materials include without limitation fibrillar crosslinkedcollagen, gelatin beads, crosslinked collagen-dPEG particles,polytetrafluoroethylene beads, silicone rubber beads, hydrogel beads,silicon carbide beads, and glass beads. Presently-preferred particulatematerials are hydroxyapatite and tricalcium phosphate.

The term "solid implant" refers to any solid object which is designedfor insertion and use within the body, and includes bone and cartilageimplants (e.g., artificial joints, retaining pins, cranial plates, andthe like, of metal, plastic and/or other materials), breast implants(e.g., silicone gel envelopes, foam forms, and the like), catheters andcannulas intended for long term (beyond about three days) use in place,artificial organs and vessels (e.g., artificial hearts, pancreases,kidneys, blood vessels, and the like), drug delivery devices (includingmonolithic implants, pumps and controlled release devices such as Alzet®minipumps, steroid pellets for anabolic growth or contraception, and thelike), sutures for dermal or internal use, periodontal membranes,ophthalmic shields, corneal lenticules, and the like.

The term "in situ" as used herein means at the place of administration.Thus, the injectable reaction mixture compositions are injected orotherwise applied to a site in need of augmentation, and allowed tocrosslink at the site of injection. Suitable sites will generally beintradermal or subcutaneous regions for augmenting dermal support, atthe site of bone fractures for wound healing and bone repair, and withinsphincter tissue for sphincter augmentation (e.g., for restoration ofcontinence).

The term "aqueous mixture" of collagen includes liquid solutions,suspension, dispersions, colloids, and the like containing collagen andwater.

The term "NFC cartilage" as used herein refers to a composition of theinvention which resembles cartilage in physical consistency. NFCcartilage is prepared from nonfibrillar collagen (e.g., collagen insolution) and is cross-linked with a hydrophillic polymer, especiallyusing dPEG. As an artifact of the production process or by design, NFCcartilage may contain about 0-20% fibrillar collagen. NFC cartilage isgenerally prepared by adding dPEG in acidic solution to an acidicsolution of collagen, and allowing conjugation to occur prior toneutralization. The term "NFC-FC cartilage" refers to a compositionsimilar to NFC cartilage, wherein the percentage of fibrillar collagenis about 20-80%. NFC-FC cartilage is generally prepared by adding dPEGin a neutralizing buffer to an acidic solution of collagen. Theneutralizing buffer causes collagen fibril formation during theconjugation process. Similarly, "FC cartilage" refers to a compositionof the invention which is prepared from fibrillar collagen and adifunctional hydrophillic polymer. FC cartilage may generally beprepared using dPEG and fibrillar collagen in neutralsolutions/suspensions.

B. General Method

B.1 Preparation

In most general terms, a suitable collagen is chemically bonded to aselected synthetic hydrophilic polymer. Suitable collagens include alltypes, preferably types I, II and III. Collagens may be soluble (forexample, commercially available Vitrogen® 100 collagen-in-solution), andmay have or omit the telopeptide regions. Preferably, the collagen willbe reconstituted fibrillar atelopeptide collagen, for example Zyderm®collagen implant (ZCI) or atelopeptide collagen in solution (CIS).Various forms of collagen are available commercially, or may be preparedby the processes described in, for example, U.S. Pat. Nos. 3,949,073;4,488,911; 4,424,208; 4,582,640; 4,642,117; 4,557,764; and 4,689,399,all incorporated all incorporated herein by reference.

The compositions of the invention comprise collagen chemicallyconjugated to a selected synthetic hydrophilic polymer or polymers.Collagen contains a number of available amino and hydroxy groups whichmay be used to bind the synthetic hydrophilic polymer. The polymer maybe bound using a "linking group", as the native hydroxy or amino groupsin collagen and in the polymer frequently require activation before theycan be linked. For example, one may employ compounds such asdicarboxylic anhydrides (e.g., glutaric or succinic anhydride) to form apolymer derivative (e.g., succinate), which may then be activated byesterification with a convenient leaving group, for example,N-hydroxysuccinimide, N,N'-disuccinimidyl oxalate, N,N'-disuccinimidylcarbonate, and the like. See also Davis, U.S. Pat. No. 4,179,337 foradditional linking groups. Presently preferred dicarboxylic anhydridesthat are used to form polymer-glutarate compositions include glutaricanhydride, adipic anhydride, 1,8-naphthalene dicarboxylic anhydride, and1,4,5,8-naphthalenetetracarboxylic dianhydride. The polymer thusactivated is then allowed to react with the collagen, forming acollagen-polymer composition of the invention.

In a preferred embodiment, monomethylpolyethylene glycol (mPEG) (mw5,000) is reacted with glutaric anhydride to form mPEG glutarate. Theglutarate derivative is then reacted with N-hydroxysuccinimide to form asuccinimidyl monomethylpolyethylene glycol glutarate. The succinimidylester (mPEG*, denoting the activated PEG intermediate) is then capableof reacting with free amino groups present on collagen (lysine residues)to form a collagen-PEG conjugate of the invention wherein one end of thePEG molecule is free or nonbound. Other polymers may be substituted forthe monomethyl PEG, as described above. Similarly, the coupling reactionmay be carried out using any known method for derivatizing proteins andsynthetic polymers. The number of available lysines conjugated may varyfrom a single residue to 100% of the lysines, preferably 10%-50%, andmore preferably 20-30%. The number of reactive lysine residues may bedetermined by standard methods, for example by reaction with TNBS.

The resulting product is a smooth, pliable, rubbery mass having a shinyappearance. It may be wetted, but is not water-soluble. It may beformulated as a suspension at any convenient concentration, preferablyabout 30-65 mg/mL, and may be implanted by injection through a suitablesyringe. The consistency of the formulation may be adjusted by varyingthe amount of liquid used.

Formulations suitable for repair of bone defects or nonunions may beprepared by providing high concentration compositions ofcollagen-polymer, or by admixture with suitable particulate materials.Such collagen-polymer particulate compositions may be malleable orrigid, depending on the amount of liquid incorporated. Formulations fortreatment of stress-bearing bone is preferably dried and rigid, and willgenerally comprise between about 45% and 85% particulate mineral, forexample hydroxyapatite or tricalcium phosphate. The tensile strength andrigidity may be further increased by heating the composition undervacuum at about 60°-90° C., preferably about 75° C., for about 5 to 15hours, preferably about 10 hours. Malleable compositions may be used forrepair of non-stressed bone.

The activated mPEG* may be replaced, in whole or in part, bydifunctional activated PEG (dPEG*, e.g., non-methylated PEG which isthen activated at each end), thus providing a crosslinked or partiallycrosslinked collagen composition. Such compositions are, however, quitedistinct from conventionally-crosslinked collagen compositions (e.g.,using heat, radiation, glutaraldehyde, glycosaminoglycans and the like),as the long-chain synthetic hydrophilic polymer imparts a substantialhydrophilic character to the composition. In a presently preferredembodiment, approximately 1∝20% of the mPEG is difunctional PEG. Thecharacter of the composition may be adjusted as desired, by varying theamount of difunctional PEG included during the process.

In another presently preferred embodiment, difunctional PEG*(substantially 100% at pH 7) is used to crosslink collagen. In oneversion, CIS (about 3-100 mg/mL, preferably about 10-40 mg/mL) isallowed to react with dPEG* (difunctional PEG activated at each end byaddition of an acid anhydride having a leaving group such assuccinimide) having a molecular weight of about 2,000 to about 20,000(preferably about 3,400-10,000) which is added as a concentratedsolution to a final reaction mixture concentration of about 5-40%,preferably about 10-20%. This represents a 5- to 10-fold excess of dPEG*to collagen on a molar basis. The collagen molecules bind to dPEG*,without mechanical mixing or agitation, and settle out of solution toproduce a cartilaginoid collagen-polymer conjugate containingapproximately 20-80% fibrillar collagen. The conjugate is then washedwith PBS to remove any remaining unreacted dPEG*, providing the materialof the invention. A cartilaginoid collagen-polymer conjugate may also beprepared by mixing dPEG* solution (pH 3) with collagen-in-solutionbetween two syringes to homogeneity, and then casting into a suitablecontainer (e.g., a Petri dish). A 20% w/v dPEG* solution (pH 7) is thenadded to the non-fibrillar collagen-PEG solution to result in a lightlycartilaginoid fibrillar collagen-polymer conjugate. The resulting NFC-FCconjugate cartilage contains approximately 1-40% fibrillar collagen. Thecharacteristics of the final product may be adjusted by varying theinitial reaction conditions. In general, increased collagen and/orpolymer concentrations provide a denser, less porous product. By varyingthe pH of the collagen solution and the dPEG* solution, compositions maybe producing over a wide range of fibrillar content. If desired, thedenser formulations may be cast or molded into any shape desired, forexample into sheets or membranes, into tubes or cylinders, into cords orropes, and the like.

A particulate microgel material may be achieved by agitating a reactionmixture of collagen and dPEG* during crosslinking (e.g., by stirring orpassing between syringes). Such materials are smooth, pliable, rubberymasses, with a shiny appearance, however, they have higher tensilestrength than collagen-mPEG conjugates or glutaraldehyde chemicallycrosslinked collagen that is not conjugated to a polymer. The injectableformulations (gels or solutions) may be used to dip coat implants,catheters, tubes (e.g., for vein replacement), meshes (e.g., for tissuereinforcement) and the like. Gels may be prepared by reducing thepolymer concentration or reducing the reaction time. CIS is thepreferred starting material where the desired properties are highdensity, rigidity, viscosity, and translucence. However, one maysubstitute fibrillar collagen (preferably atelopeptide fibrillarcollagen such as ZCI) and obtain products which are more opaque, moreflexible, and more susceptible to colonization by cells afterimplantation. CIS-based materials are presently preferred for coatingarticles to be implanted, such as catheters and stress-bearing boneimplants. Fibrillar collagen-based materials are preferred forapplications such as dermal augmentation, sphincter augmentation,resurfacing of eroded joint surfaces (as in rheumatoid arthritis),replacement of tendons and ligaments, and preparation of artificialvessels (e.g., veins).

Compositions of the invention containing biological growth factors suchas EGF and TGF-β are prepared by mixing an appropriate amount of thefactor into the composition, or by incorporating the factor into thecollagen prior to treatment with activated PEG. By employing anappropriate amount of difunctional PEG, a degree of crosslinking may beestablished, along with molecules consisting of collagen linked to afactor by a synthetic hydrophilic polymer Preferably, the factor isfirst reacted with a molar excess of dPEG* in a dilute solution over a 3to 4 hour period. The factor is preferably provided at a concentrationof about 1 μg/mL to about 5 mg/mL, while the dPEG* is preferably addedto a final concentration providing a 30 to 50-fold molar excess. Theresulting conjugated factor is then added to an aqueous collagen mixture(about 1 to about 60 mg/mL) at pH 7-8 and allowed to react further. Theresulting composition is allowed to stand overnight at ambienttemperature. The pellet is collected by centrifugation, and is washedwith PBS by vigorous vortexing in order to remove non-bound factor.

Flexible sheets or membranous forms of the collagen-polymer conjugatemay be prepared by methods known in the art, for example, U.S. Pat. Nos.4,600,533; 4,412,947; and 4,242,291. Briefly, high concentration (10-100mg/mL) CIS or fibrillar collagen (preferably atelopeptide fibrillarcollagen, such as ZCI) is cast into a flat sheet container. A solutionof mPEG* (having a molecular weight of approximately 5,000) is added tothe cast collagen solution, and allowed to react overnight at roomtemperature. The resulting collagen-polymer conjugate is removed fromthe reaction solution using a sterile spatula or the like, and washedwith PBS to remove excess unreacted mPEG*.

The resulting conjugate may then be compressed under constant pressureto form a uniform, flat sheet or mat, which is then dried to form amembranous implant of the invention. More flexible membranous forms areachieved by using lower collagen concentrations and high polymerconcentrations as starting materials.

Less flexible membranous forms are prepared by using a dPEG* solutionrather than mPEG*. CIS, at room temperature, is mixed with a buffersolution and incubated at 37° C. overnight. The resulting gel iscompressed under constant pressure, dried, and desalted by washing. Theresultant membrane is then crosslinked by treating with dPEG*, washed,and then dried at low temperature.

Collagen-polymer conjugates may also be prepared in the form of sponges,by lyophilizing an aqueous slurry of the composition after conjugation.

Alternatively, CIS or fibrillar collagen (10-100 mg/mL) is cast into aflat sheet container. A solution of dPEG* (22-50% w/v) is added to thecast collagen The mixture is allowed to react over several hours at roomtemperature. Shorter reaction times result in more flexible membranes.The resulting collagen-polymer membrane may be optionally dehydratedunder a vacuum oven, lyophilization, or air-drying.

B.2 Use and Administration

Compositions of the invention have a variety of uses. Malleable, plasticcompositions may be prepared as injectable formulations, and aresuitable for dermal augmentation, for example for filling in dermalcreases, and providing support for skin surfaces. Such compositions arealso useful for augmenting sphincter tissue, (e.g., for restoration ofcontinence). In such cases, the formulation may be injected directlyinto the sphincter tissue to increase bulk and permit the occludingtissues to meet more easily and efficiently. These compositions may behomogeneous, or may be prepared as suspensions of small microgelcollagen-polymer conjugate particles or beads.

Surprisingly, one may administer the reaction mixture by injectionbefore crosslinking has completed. In this embodiment, an aqueouscollagen mixture is combined with a low-concentration dPEG* solution,mixed, and the combination injected or applied before the viscosityincreases sufficiently to render injection difficult (usually about 20minutes). Mixing may be accomplished by passing the mixture between twosyringes equipped with Luer lock hubs, or through a single syringehaving dual compartments (e.g., double barrel) The compositioncrosslinks in situ, and may additionally crosslink to the endogenoustissue, anchoring the implant in place. In this method, one can usecollagen (preferably fibrillar collagen) at a concentration of about10-100 mg/mL, although abut 30-80 mg/mL is preferred, most preferablyabout 33 mg/mL. The dPEG* concentration is preferably set at about 0.1to about 3%, although concentrations as high as 30% may be used ifdesired. The mixture is injected directly into the site in need ofaugmentation, and causes essentially no detectable inflammation orforeign body reaction. One may additionally include particulatematerials in the collagen reaction mixture, for example hydrogel orcollagen-dPEG beads, or hydroxyapatite/tricalcium phosphate particles,to provide a bulkier or more rigid implant after crosslinking.

Compositions of the invention (particularly cross-linked collagencompositions) are also useful for coating articles for implantation orrelatively long term residence within the body. Such surface treatmentrenders the object nonimmunogenic, and reduces the incidence of foreignbody reactions. Accordingly, one can apply compositions of the inventionto catheters, cannulas, bone prostheses, cartilage replacement, breastimplants, minipumps and other drug delivery devices, artificial organs,and the like. Application may be accomplished by dipping the object intothe reaction mixture while crosslinking is occurring, and allowing theadherent viscous coating to dry. One may pour or otherwise apply thereaction mixture if dipping is not convenient. Alternatively, one mayuse flexible sheets or membranous forms of collagen-polymer conjugate towrap the object with, sealing corners and edges with reaction mixture.

In another embodiment, the object may be dipped in a viscouscollagen-in-solution bath, or in a fibrillar collagen solution until theobject is completely coated. The collagen solution is fixed to theobject by dipping the collagen-coated object into a dPEG* (pH 7)solution bath, and then allowing the collagen-polymer coated object todry. Alternatively, viscous collagen-in-solution is mixed with a dPEG*(pH 3) solution and polymerized rapidly, as described above. The objectis dipped in the acidic collagen-polymer solution, and cured by dippingthe coated object into a neutralizing buffer containing about 20% byweight dPEG* (pH 7), to result in a collagen-polymer coated object.

Compositions of the invention may be prepared in a form that is denseand rigid enough to substitute for cartilage. These compositions areuseful for repairing and supporting tissue which require some degree ofstructure, for example in reconstruction of the nose, ear, knee, larynx,tracheal rings, and joint surfaces. One can also replace tendon,ligament and blood vessel tissue using appropriately formedcartilaginoid material. In these applications, the material is generallycast or molded into shape: in the case of tendons and ligaments, it maybe preferable to form filaments for weaving into cords or ropes. In thecase of artificial blood vessels it may be advantageous to incorporate areinforcing mesh (e.g., nylon or the like).

Compositions of the invention which contain growth factors areparticularly suited for sustained administration of factors, as in thecase of wound healing promotion. Osteoinductive factors and cofactors(including TGF-β) may advantageously be incorporated into compositionsdestined for bone replacement, augmentation, and/or defect repair.Compositions provided in the form of a membrane may be used to wrap orcoat transplanted organs, to suppress rejection and induce improvedtissue growth. Similarly, one may dip coat organs for transplantationusing a crosslinking reaction mixture of factor-polymer conjugates andcollagen Alternatively, one may administer antiviral and antitumorfactors such as TNF, interferons, CSFs, TGF-β, and the like for theirpharmaceutical activities. The amount of composition used will dependupon the severity of the condition being treated, the amount of factorincorporated in the composition, the rate of delivery desired, and thelike. However, these parameters may easily be determined by routineexperimentation, for example by preparing a model composition followingthe examples below, and assaying the release rate in a suitable animalmodel.

C. Examples

The examples presented below are provided as a further guide to thepractitioner of ordinary skill in the art, and are not to be construedas limiting the invention in any way.

EXAMPLE 1 (Preparation of Collagen-PEG)

(A) Monomethyl-PEG 5000 (50 g, 10 mmol, Aldrich Chemical Co.) isdissolved in 1,2-dichoroethane (250 mL) and heated at reflux withglutaric anhydride (5 g) and pyridine (4 mL) under nitrogen for 3 days.The solution is then filtered and the solvent evaporated, and theresidue dissolved in water (100 mL) and washed with diethyl ether (2×50mL). The resulting PEG-glutarate is extracted from the water withchloroform (2×50 mL), and the chloroform evaporated to yield about 43 gof PEG-glutarate. The PEG-glutarate is then dissolved indimethylformamide (DMF, 200 mL) at 37° C., and N-hydroxysuccinimide (10%molar xs) added. The solution is cooled to 0° C., and an equivalentamount of dicyclohexylcarbodiimide added in DMF solution (10 mL). Themixture is left at room temperature for 24 hours, and then filtered.Cold benzene (100 mL) is then added, and the PEG-succinimidyl glutarate(PEG-SG) precipitated by adding petroleum ether (200 mL) at 0° C. Theprecipitate is collected on a sintered glass filter. Dissolution inbenzene, followed by precipitation with petroleum ether is repeatedthree times to provide "activated" PEG (PEG-SG).

Vitrogen 100® collagen in solution (400 mL, 1.2 g collagen, 0.004 mmol)was mixed with 0.2 M phosphate buffer (44 mL) to elevate the pH to 7.4.Next, a three-fold molar excess of PEG-SG (6.00 g, 1.2 mmol) wasdissolved in water for injection (40 mL) and sterile-filtered. ThePEG-SG solution was then added to the collagen solution, and the mixtureallowed to stand at 17°-22° C. for about 15 hours. The solution was thencentrifuged, and the resulting pellet (25 g) of reconstituted fibrilscollected and washed with phosphate-buffered saline (PBS, 3×400 mL) toremove residual PEG. The resulting material has a solid, coherentelasticity, and may be picked up on a spatula (the equivalentnon-conjugated collagen, Zyderm® collagen implant is more fluid). Theresulting material may be diluted with PBS to provide a dispersionhaving 20.5 mg/mL collagen-PEG.

(B) Similarly, proceeding as in part (A) above but substitutingpolypropylene glycol and POE-POP block polymers for polyethylene glycol,the corresponding collagen-PPG and collagen-POE-POP compositions areprepared.

(C) Difunctional PEG 3400 (34 g, 10 mmol, Aldrich Chemical Co.) isdissolved in 1,2-dichoroethane (250 mL) and heated at reflux withglutaric anhydride (10 g) and pyridine (4 mL) under nitrogen for 3 days.The solution is then filtered and the solvent evaporated, and theresidue dissolved in water (100 mL) and washed with diethyl ether (2×50mL). The resulting PEG-diglutarate is extracted from the water withchloroform (2×50 mL), and the chloroform evaporated to yieldPEG-diglutarate. The PEG-diglutarate is then dissolved in DMF (200 mL)at 37° C., and N-hydroxysuccinimide (10% molar xs) added. The solutionis cooled to 0° C., and an equivalent amount of dicyclohexylcarbodiimideadded in DMF solution (-0 mL). The mixture is left at room temperaturefor 24 hours, and then filtered. Cold benzene (100 mL) is then added,and the PEG-di(succinimidyl glutarate) (dPEG-SG) precipitated by addingpetroleum ether (200 mL) at 0° C. The precipitate is collected on asintered glass filter. Dissolution in benzene, followed by precipitationwith petroleum ether is repeated three times to provide "activated" dPEG(dPEG*).

Vitrogen 100® collagen in solution (400 mL, 1.2 g collagen, 0.004 mmol)was mixed with 0.2 M phosphate buffer (44 mL) to elevate the pH to 7.4.Next, a three-fold molar excess of dPEG* (6.00 g, 1.2 mmol) wasdissolved in water for injection (40 mL) and sterile-filtered. The dPEG*solution was then added to the collagen solution, agitated, and themixture allowed to stand at 17°-22° C. for about 15 hours. The solutionwas then centrifuged, and the resulting pellet of reconstituted fibrilscollected and washed with PBS (3×400 mL) to remove residual dPEG*. Thepellet was then placed in a syringe fitted with a Luer lock hubconnected to a second syringe, and was passed between the syringes untilhomogeneous. The resulting material is a microgel or a particulatesuspension of random size fibrils in solution (microgel conjugate). Thematerial is a smooth, pliable, rubbery mass, with a shiny appearance.

(D) Preparation of Cartilaginoid Conjugates:

Approximately 20% by weight of dPEG* (pH 7) was added to collagen insolution (33.8 mg/mL), and incubated at 21° C. for about 16 hours. Theresulting conjugate was washed with 100 mL PBS 3-5 times over 12 hours.The resulting cartilaginoid non-fibrillar collagen-polymer conjugate(NFC-FC cartilage) was a translucent solid with coherent elasticity. Theproduct contained approximately 20-80% fibrillar collagen.

Another NFC cartilage composition was prepared by mixing dPEG* solution(0.6 g, pH 3) with collagen in solution (33.8 mg/mL, pH 2). The mixturewas passed between two syringes joined by a Luer lock connector to forma homogenous solution. A solution of dPEG* (20% w/v) in a neutralizingbuffer was then added to result in a substantially non-fibrillarcollagen (NFC) cartilage material. The resulting product containedapproximately 1-40% fibrillar collagen.

Alternatively, fibrillar collagen may be used instead of CIS to producea cartilaginoid fibrillar collagen-polymer conjugate (FC cartilage)having an opaque appearance and high fibrillar content. Such FCcartilage is more porous and permeable than non-fibrillarcollagen-polymer conjugates.

EXAMPLE 2 (Characterization)

(A) Collagen-mPEG prepared in Example IA was characterized and comparedwith Zyderm® collagen implant (ZCI), and glutaraldehyde-crosslinkedfibrillar collagen (GAX).

Extrusion

This assay measured the force required to extrude the test compositionthrough a 30 gauge needle. The results are shown in FIG. 1. As can beseen from the graph of force required (in Newtons) versus plungertravel, ZCI was extruded smoothly, requiring a force of about 20-30Newtons. GAX was not extruded smoothly, as shown by the "spiking"exhibited in the force trace. At the plateau, GAX required about 10-15 Nfor extrusion. In contrast, collagen-mPEG demonstrated a very lowextrusion force (8-10 N), with little or no spiking.

Intrusion

Intrusion is a measure of the tendency of a composition to "finger" orchannel into a porous bed, rather than remaining in a compact mass. Lowintrusion is preferred in augmentation of soft tissue, so that theinjected implant does not diffuse through the dermis and remains inplace.

A 1 mL syringe fitted with a 30 gauge needle was half-filled withsilicon carbide particles (60 mesh), simulating human dermis. The upperhalf of the syringe was filled with 0.5 mL test composition (GAX, ZCI,or collagen-mPEG) at 35 mg/mL. The plunger was then fitted, anddepressed. On depression, ZCI appeared at the needle, demonstratingintrusion through the silicon carbide bed. Syringes filled with GAX orcollagen-mPEG of the invention did not pass collagen, instead releasingonly buffer, demonstrating no intrudability.

Helicity

The portion of each composition exhibiting nonhelical character wasmeasured using sensitivity to digestion with trypsin. Samples weretreated with the protease trypsin, which is capable of attacking onlyfragmented portions of the collagen protein. The extent of hydrolysis ismeasured by fluorescamine assay for solubilized peptides, and theresults are expressed as percentage non-helical collagen. The percentageof non-helical collagen was measured 30 minutes after the beginning ofthe digestion period. The results indicated that ZCI was 3-10%sensitive, GAX was 1-2% sensitive, and collagen-mPEG was about 1%sensitive. Sensitivity to trypsin may also correlate to sensitivity toendogenous proteases following implantation.

Collagenase Sensitivity

The sensitivity of each composition to collagenase ZCI was 65.2%digested, compared to 2.2% GAX, and 45.8% for collagen-mPEG.

Phase Transition

The behavior of each composition vs. temperature was examined using adifferential scanning calorimeter. On heating, ZCI exhibited multiplepeaks at about 45 and 53.C. GAX exhibited a peak at 67°-70° C.Collagen-mPEG exhibited a peak at 56°-61° C.

Lysine Content

The number of free lysines per mole was determined for each compositionusing TNBS to quantify reactive epsilon amino groups. ZCI exhibitedabout 30 lysines per (single helix) molecule (K/m), whereas GAXexhibited 26-27 K/m, and collagen-mPEG 21-26 K/m.

(B) Characterization of Crosslinked Collagen-Polymer Conjugates

A collagen-dPEG conjugate prepared as described in was characterizedusing differential scanning calorimetry (DSC). This test is a measure ofthe transition temperature during fragmentation of the collagen moleculeat a microscopic level. A lowering of the transition temperatureindicates an increase in fragmentation in a manner similar to thatmeasured by trypsin sensitivity.

The collagen-dPEG conjugate showed a single denaturational transition at56° C. by DSC, which is similar to the typical melting point of theCollagen-PEG conjugate prepared in Example IA. In comparison, ZCI has amelting temperature of 45°-53° C. with multiple denaturationaltransitions, and GAX has a melting temperature of 67°-70° C. with asingle denaturational transition.

The extrusion test described in Example 2A could not be used tocharacterize the collagen-dPEG conjugate because the material was notextrudable through a 30 gauge needle.

Using the intrusion test described in Example 2A, the passage ofcollagen-dPEG was completely blocked at the silicon carbide bed, whichindicates high crosslinking between the collagen molecules and little orno intrudability.

EXAMPLE 3 (Immunogenicity)

(A) Non-crosslinked PEG-Collagen

This experiment was conducted to demonstrate the relative immunogenicityof a collagen-mPEG preparation of the invention versus acommercially-available bovine collagen formulation prepared fromessentially the same source material, and having a similar consistency.As both collagen preparation were prepared using atelopeptide collagen(which is only weakly immunogenic), the preparations were formulatedwith either complete Freund's adjuvant (CFA) or incomplete Freund'sadjuvant (IFA), to enhance the immune response. This is a severe test,designed to magnify any possible immune reaction.

Collagen-mPEG was prepared as in Example IA above. Male Hartley guineapigs (11) were anesthetized and bled by heart puncture forpre-immunization serologic evaluation. Five animals were treated withtwo 0.1 mL intramuscular injections of Zyderm® collagen implant (ZCI)emulsified in CFA (1:9) in the left and right thighs. Another fiveanimals were treated in the same fashion, using collagen-PEG (35 mg/mL)emulsified in CFA. One animal was treated with collagen-PEG in IFA. Atday 14 following immunization, all animals were again bled by heartpuncture, and serum obtained for antibody titer determination (usingELISA). Serology was again performed at day 30.

On day 30, following collection of serum samples, each animal waschallenged intradermally with both ZCI and collagen-PEG (0.1 mL of each,one on each flank). Delayed-type hypersensitivity (DTH) was quantifiedas a measure of cell-mediated immunity. DTH was evaluated at 24, 48, and72 hours post-challenge by measuring the diameter of any wheal usingmicrometer calipers, and noting the extent of erythema and induration.Animals were then euthanized with CO₂, and the injection sites excisedand fixed in neutral, buffered formalin for histological study.

Serological results indicated reduced immunogenicity of collagen-PEG vs.ZCI. At day 14, 80% of ZCI immunized animals exhibited "positive"antibody responses (titer≧160 at day 14), whereas 0% of the collagen-PEGimmunized animals exhibited positive responses. At day 30, allZCI-immunized animals exhibited high antibody titers, whereas none ofthe collagen-PEG-immunized animals (C-PEG) exhibited high titers. Thedata are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Immunogenicity                                                                                  Antibody Titer                                              Animal    Treatment     day 14  day 30                                        ______________________________________                                        1         ZCI           320     >2560                                         2         ZCI           320     1280                                          3         ZCI           2560    >2560                                         4         ZCI           320     >2560                                         5         ZCI            80     2560                                          6         C-PEG          0        0                                           7         C-PEG          0       160                                          8         C-PEG          40      640                                          9         C-PEG          0        20                                          10        C-PEG          0       640                                          11        C-PEG (IFA)    0       160                                          ______________________________________                                    

Responses to the DTH challenge also demonstrated that the collagen-mPEGof the invention is less immunogenic. Guinea pigs immunized with ZCI andchallenged with ZCI exhibited a wheal measuring 1.128±0.058 cm indiameter. Animals immunized with collagen-mPEG and challenged withcollagen-mPEG exhibited wheals measuring 0.768±0.036 cm. Animalsimmunized with ZCI and challenged with collagen-mPEG, or immunized withcollagen-mPEG and challenged with ZCI, developed wheals smaller than theZCI-immunized ZCI-challenged wheals. Responses measured at 48 and 72hours were essentially the same or lower than the 24 hour response foreach site. Erythema was essentially the same for all animals.

Histological studies showed that both materials exhibited comparableintrusion, fingering into the dermis and subcutaneous space. Sites ofintradermal challenge with ZCI in ZCI-immunized animals exhibited themost extensive inflammatory response, including a cellular infiltrate oflymphohistiocytic elements with eosinophils and occasional giant cells.Two of the implant sites demonstrated an erosive inflammation of theoverlying epidermis and eschar formation. Sites of intradermal challengewith collagen-mPEG in ZCI-immunized animals exhibited only a moderateassociated inflammatory infiltrate, with a marked reduction in acutecells and lymphoid elements. Histiocytes and giant cells were moreprevalent, and in some samples lined and colonized the implants heavily.Animals immunized with collagen-mPEG exhibited only slight to moderatereaction, with ZCI challenge sites accompanied by a modestlymphohistiocytic perivascular infiltrate with a few eosinophils andgiant cells. Collagen-mPEG challenge sites were typically accompanied bya minimal scattering of lymphoid cells near the associated vasculature.

(B) Crosslinked dPEG-Collagen Conjugates

Collagen-dPEG conjugates were prepared as in Example ID. The sampleswere implanted in the dorsal subcutis and as cranial onlays in rats.After implantation for 30 days in the subcutis, NFC cartilage and NFC-FCcartilage materials had a homogeneous microfibrillar structure. Mildcolonization by connective tissue cells occurred at the periphery of theNFC-FC cartilage samples, and mild capsule formation was present. Nocolonization had occurred with the NFC cartilage material and mildcapsule formation was present. FC cartilage had a very fibrous structurewith mild but frequently deep colonization by connective tissue cellsand sparse numbers of adipocytes. Trace amounts of capsule were presentin limited areas of the FC cartilage samples. NFC cartilage materialstended to retain their pre-implantation shape, with sharply definededges, while the NFC-FC cartilage samples tended to flatten over timeand develop rounded profiles.

When implanted as cranial onlays, the appearance of each of thematerials was similar to that in the subcutis except that the samplestended to become anchored to the skull via integration of the capsule orsurrounding loose connective tissue with the periosteum.

All of the samples appeared to be biocompatible, have differing degreesof colonization by host tissues, and varying mechanical characteristics.

EXAMPLE 4 (In situ Crosslinking)

A dPEG solution was prepared as described in Example 1C above. Thefollowing samples were then prepared:

(1) 5 mg dPEG in 80 μL water, mixed with 0.5 mL fibrillar collagen (35mg/mL), to a final dPEG concentration of 1% by volume;

(2) 15 mg dPEG in 80 μL water, mixed with 0.5 mL fibrillar collagen (35mg/mL), to a final dPEG concentration of 3% by volume;

(3) Vitrogen® 100 collagen in solution;

(4) 5 mg dPEG in 80 μL water, mixed with 0.5 mL non-fibrillar collagen(35 mg/mL), to a final dPEG concentration of 1% by volume;

(5) 15 mg dPEG in 80 μL water, mixed with 0.5 mL nonfibrillar collagen(35 mg/mL), to a final dPEG concentration of 3% by volume;

(6) 5 mg dPEG in 0.5 ml PBS, to a final dPEG concentration of 1% byvolume; and

(7) GAX.

The dPEG solutions of Samples 1, 2, 4, and 5 were placed in a 1 mLsyringe equipped with a Luer lock fitting and connector, and joined toanother syringe containing the collagen material. The solutions weremixed by passing the liquids back and forth between the syringes severaltimes to form the homogeneous reaction mixture.

The syringe connector was then removed and replaced with a 27 gaugeneedle, and approximately 50 μL of the reaction mixture was injectedintradermally into each of 20 guinea pigs. Samples 3, 6, and 7 weresimilarly administered through a 27 gauge needle. At intervals up to 30days following injection, the treatment sites were harvested and studiedhistologically.

By 30 days, all of the materials appeared to be biocompatible. Samples 1and 2 displayed wide dispersion with an intermediate degree ofinterdigitation with dermal collagen fibers. Colonization by connectivetissue cells was moderate, and a trace of round cell infiltrate witheosinophils was seen.

Samples 3, 4 and 5 were highly dispersed and finely interdigitated withdermal collagen fibers. Colonization was mild to moderate, and tracelevels of round cell infiltration were seen.

Sample 6 had no detectable effects. Sample 7 occurred as large islandswith moderate colonization and trace to mild levels of inflammation.

EXAMPLE 5 (Coating of Implants)

A collagen-dPEG reaction mixture was prepared as described in Example 1Cabove. A titanium implant was dipped into the reaction mixtureapproximately 20 minutes after cross-linking was initiated. The implantwas then allowed to finish crosslinking, and dry overnight.

EXAMPLE 6 (Collagen-Polymer-Growth Factor Conjugates)

(A) A conjugate containing crosslinked collagen-dPEG-TGF-β1 was preparedas follows:

A solution of TGF-β1 and ¹²⁵ I-TGF-β1 (10⁵ cpm; 25 μL of 1 mg/mL) wasadded to a solution of dPEG* (4 mg) in CH₂ Cl₂ (100 μL), and the mixtureallowed to react for 12 (sample #3) or 35 (sample #5) minutes at 17° C.To this was added 2.5 mL of collagen solution (3 mg/mL atelopeptidenonfibrillar collagen), and the resulting mixture allowed to incubateovernight at ambient temperature. The pellet which formed was collectedby centrifugation to provide collagen-dPEG-TGF-β1.

(B) A composition based on fibrillar atelopeptide collagen was preparedas in part A above, but limiting TGF-β1/dPEG* reaction time to 2minutes, and substituting 7 mg of fibrillar collagen (precipitated fromcollagen in solution within 2 minutes prior to use) for collagen insolution.

(C) A composition containing dPEG-crosslinked collagen and free TGF-β1was prepared as follows:

A solution of dPEG* (4 mg) in CH₂ Cl₂ (100 μL), was added to 2.5 mL ofCIS (3 mg/mL atelopeptide nonfibrillar collagen), and the resultingmixture allowed to incubate overnight at ambient temperature. The pelletwhich formed was washed to remove unreacted dPEG*, and 25 μg of TGF-β1mixed in to provide collagen-dPEG+TGF-β1.

(D) The degree of TGF-β1 binding was determined as follows:

Each composition prepared in parts A-C above was washed six times with0.5 mL of buffer (0.02 M phosphate buffer, 0.1% BSA) by vigorousvortexing followed by centrifugation in order to remove non-boundTGF-β1. The pellet and supernatants were collected at each time ofwashing, and were counted. FIG. 2 demonstrates the release rate of thecompositions of part A (open circles) and part B (filled circles) versusthe simple mixture prepared in part C (x's), showing the number ofcounts released as a function wash cycle. As shown in the figure, theTGF-β1 in the simple mixture is quantitatively released within about 6washings, while approximately 40% of the TGF-β1 is retained in thecompositions of part B and 50% is retained in the compositions of partA.

(E) The biological activity of the materials prepared above was assayedas follows:

Compositions prepared according to part A (CIS-dPEG-TGF-β1)(TGF-β1/dPEG* reaction time of 12 minutes) and part C (CIS-dPEG+TGF-β1)were prepared, as well as a control prepared according to part C withoutTGF-β1 (CIS-dPEG). The samples were washed in PBS/BSA eight times asdescribed in part D, then washed an additional three times in fetalbovine serum (Gibco) at 37° C. This washing protocol resulted invisually detectable material loss, so remaining TGF-β1 content wasdetermined by counting the remaining ¹²⁵ I. TGF-β1 activity was thenassayed by ELISA. The results are shown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Retention of Biological Activity                                                            125.sub.I remaining  O.D.                                       Sample        Counts    TGF-β1 (μg)                                                                      (414 nm)                                   ______________________________________                                        CIS-dPEG        0       0          0.015                                                                         0.015                                      CIS-dPEG + TGF-β1                                                                       2775     0.5-1.0    0.029                                                                         0.035                                      CIS-dPEG-TGF-β1                                                                        42604     7.4        0.102                                                                         0.082                                      ______________________________________                                    

The data demonstrates that the TGF-β1 retained in the compositions ofthe invention remains in a substantially active form.

EXAMPLE 7 (Formulations)

(A) A formulation suitable for implantation by injection was prepared bysuspending collagen-PEG in sterile water for injection, at 35 mg/mL. Thecharacteristics of the resulting formulation are described in Example 2above.

(B) A formulation useful for repair of stress-bearing bone defects(e.g., fractures, nonunions, and the like) may be prepared by mixingcollagen-PEG of the invention with a suitable particulate, insolublecomponent. The insoluble component may be fibrillar crosslinkedcollagen, gelatin beads, polytetrafluoroethylene beads, silicone rubberbeads, hydrogel beads, silicon carbide beads, mineral beads, or glassbeads, and is preferably a calcium mineral, for example hydroxyapatiteand/or tricalcium phosphate.

Solid formulations were prepared by mixing Zyderm® II (65 mg/mLcollagen) or collagen-mPEG (63 mg/mL) with particulate hydroxyapatiteand tricalcium phosphate (HA+TCP) and air drying to form a solid blockcontaining 65% HA by weight. Optionally, blocks were heat-treated byheating at 75° C. for 10 hours. The resulting blocks were hydrated in0.13 M saline for 12 hours prior to testing.

On standing, it was observed that Zyderm® -HA+TCP (Z-HA) compositionsseparated into three phases, whereas PEG-collagen-HA+TCP (PC-HA)compositions remained single phase.

Each block was elongated by 5%, and its stress relaxation monitored for1 minute after release. After this test, each block was subjected toconstant elongation at a constant 1 cm/min until failure. The resultsare shown in Table 3:

                  TABLE 3                                                         ______________________________________                                        Mechanical Strength                                                           Stress Relaxation    Constant Extension                                              Peak    Constant t.sub.1/2                                                                            Rupture                                                                              Extension                               Sample Force   Force    (min)  Force  at Rupture                              ______________________________________                                        Z-HA   1.5     1.1      0.04   2.6    11.0%                                   (air)  --      --       --     2.6    15.3%                                   Z-HA   1.5     1.1      0.06   --     --                                      (heat) 1.4     1.0      0.07   3.4    14.0%                                   PC-HA  2.6     1.8      0.06   5.5    12.3%                                   (air)  2.8     2.1      0.08   5.4    11.7%                                   PC-HA  3.3     2.6      0.04   5.4    12.0%                                   (heat) 3.6     2.7      0.06   5.4    20.3%                                   ______________________________________                                         All forces reported in newtons. Extension at rupture (strain) reported in     percent extension.                                                       

The data demonstrate that collagen-polymer forms HA+TCP compositionsexhibiting substantially greater tensile strength. Thus, one can prepareimplant compositions with collagen-polymer which are substantiallystronger than compositions employing the same amount of non-conjugatedcollagen, or may reduce the amount of collagen-polymer employed to forma composition of equal strength.

What is claimed:
 1. A pharmaceutically acceptable, non-immunogeniccomposition comprising atelopeptide collagen chemically conjugated to asynthetic hydrophilic polymer.
 2. The composition of claim 1 wherein theatelopeptide collagen is selected from the group consisting of type I,type II and type III.
 3. The composition of claim 1 wherein said polymeris polyethylene glycol having a weight average molecular weight of about400 to about 20,000.
 4. The composition of claim 1 wherein saidsynthetic hydrophilic polymer is bound to an available lysine residue onsaid collagen.
 5. The composition of claim 4 wherein said polymer has afirst end and a second end, said first end being bound to said lysineresidue, and said second end being non-bound.
 6. The composition ofclaim 5 wherein synthetic hydrophilic polymer molecules are bound to10-50% of said available lysine residues.
 7. The composition of claim 6wherein said synthetic hydrophilic polymer molecules are bound to 20-30%of said available lysine residues.
 8. The composition of claim 4 whereinsaid polymer has a first end and a second end, wherein said first andsecond ends are bound to collagen.
 9. The composition of claim 8 whereinthe ratio of collagen molecules to polymer molecules is about 1:1 toabout 1:20.
 10. The composition of claim 8 wherein said collagen isatelopeptide fibrillar collagen or nonfibrillar collagen.
 11. Thecomposition of claim 1, wherein the synthetic hydrophilic polymer ispolyethylene glycol.
 12. The composition of claim 11 wherein thecomposition is a pharmaceutically acceptable injectable composition. 13.The composition of claim 1, wherein the synthetic hydrophilic polymer ispolyethylene glycol having a molecular weight in the range of about 400to about 20,000 and the composition is a pharmaceutically acceptableinjectable composition.
 14. A composition comprising:collagen chemicallyconjugated to a synthetic hydrophilic polymer having a first end and asecond end, said first end being bound to a collagen molecule, and saidsecond end being bound to a growth factor.
 15. The composition of claim14 wherein said growth factor is selected from the group consisting ofepidermal growth factor, transforming growth factor-α, transforminggrowth factor-β, transforming growth factor-β1, transforming growthfactor-β2, platelet-derived growth factor-AA, platelet-derived growthfactor-AB, platelet-derived growth factor-BB, acidic fibroblast growthfactor, basic fibroblast growth factor, connective tissue activatingpeptide, β-thromboglobulin, insulin-like growth factors, tumor necrosisfactor, interleukins, colony stimulating factors, erythropoietin, nervegrowth factor, interferons, and osteogenic factors.
 16. The compositionof claim 15 wherein said growth factor is selected from the groupconsisting of transforming growth factor-β, transforming growthfactor-β1, transforming growth factor-β2, and erythropoietin.
 17. Apharmaceutically acceptable, non-immunogenic composition suitable foraugmentation of soft tissue, which comprises:atelopeptide collagenchemically conjugated to a synthetic hydrophilic polymer; and asufficient amount of a fluid pharmaceutically acceptable carrier. 18.The composition of claim 17 wherein the polymer is polyethylene glycoland the collagen is selected from the group consisting of non-fibrillarcollagen and fibrillar collagen.
 19. The composition of claim 17 whereinsaid polymer is monomethyl-polyethylene glycol having a weight averagemolecular weight of about 1,900 to about 8,000.
 20. The composition ofclaim 19 wherein said monomethyl-polyethylene glycol is chemicallyconjugated to 10-50% of the available lysine residues on said collagen.21. The composition of claim 17 which further comprises a tissuegrowth-promoting amount of epidermal growth factor, transforming growthfactor-β, an osteoinductive factor, or a hematopoietic factor.
 22. Thecomposition of claim 17 wherein said polymer has a first end and asecond end, wherein said first and second ends are bound to collagen toform a crosslinked collagen-polymer conjugate, said composition havingthe form of a suspension of collagen-polymer conjugate particles in apharmaceutically acceptable carrier.
 23. The composition of claim 22which further comprises a tissue growth-promoting amount of a growthfactor selected from the group consisting of epidermal growth factor,transforming growth factor-β, or a hematopoietic factor.
 24. Thecomposition of claim 23 wherein said factor is chemically bound to saidcollagen by a covalent bond between said factor and the first end ofsaid polymer, and a covalent bond between the second end of said polymerand said collagen.
 25. The composition of claim 17 wherein said fluidcarrier is present in an amount sufficient to render the compositionmalleable.
 26. The composition of claim 17 wherein said fluid carrier ispresent in an amount sufficient to render the composition injectable.27. A pharmaceutically acceptable, non-immunogenic, injectablecomposition suitable for augmentation of soft tissue, whichcomprises:atelopeptide collagen; a synthetic hydrophilic polymer havinga first and second end, wherein said first and second ends comprisereactive groups capable of forming a covalent bond in situ with anavailable lysine side chain present in collagen; and a fluidpharmaceutically acceptable carrier in an amount sufficient to form aninjectable composition.
 28. The composition of claim 27 wherein saidpolymer comprises polyethylene glycol having a weight average molecularweight of about 400 to about 20,000.
 29. The composition of claim 28wherein said reactive groups comprise N-hydroxysuccinimide esters. 30.The composition of claim 27 wherein said composition comprises about 10to about 100 mg/mL collagen and about 0.1 to about 30% synthetichydrophilic polymer having reactive groups.
 31. The composition of claim30 wherein said composition comprises about 30 to about 80 mg/mLcollagen and about 0.3 to about 10% synthetic hydrophilic polymer havingreactive groups.
 32. The composition of claim 27 wherein said collagencomprises fibrillar atelopeptide collagen.
 33. The composition of claim27 wherein said collagen comprises nonfibrillar collagen.